Identifying a de novo fetal mutation from a maternal biological sample

Abstract

Systems and methods for identifying a de novo mutation in a genome of a fetus are provided. Methods may include identifying a location of each of a plurality of cell-free nucleic acid molecules using sequence reads. Methods may also include identifying a first sequence in the sequence reads at a first location that is not present in the maternal or paternal sequences. Methods may additionally include determining a first fractional concentration of the first sequence in the biological sample at the first location. Further, methods may include determining a second fractional concentration of a fetal-specific second sequence. The second sequence may be inherited by the fetus from the father at the second location. In addition, methods may include classifying the first sequence as a de novo mutation at the first location in a fetal genome of the fetus if the first and second fractional concentrations are about the same.

What is claimed is: 
     
       1. A method of identifying a de novo mutation in a genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, the father having a paternal genome and the mother having a maternal genome, the method comprising:
 performing a random sequencing of a plurality of cell-free nucleic acid molecules from a biological sample obtained from the pregnant female to obtain sequence reads of the plurality of cell-free nucleic acid molecules; 
 receiving the sequence reads at a computer system; 
 identifying, by the computer system, a location of each of the plurality of cell-free nucleic acid molecules in a human genome using the sequence reads of the plurality of cell-free nucleic acid molecules, wherein identifying the location of a cell-free nucleic acid molecule in the human genome includes mapping at least one sequence of the cell-free nucleic acid molecule to the human genome; 
 for each of at least a portion of the locations, determining, by the computer system, one or more maternal sequences in the maternal genome and one or more paternal sequences in the paternal genome at the location; 
 identifying, by the computer system, a first sequence in the sequence reads of the plurality of cell-free nucleic acid molecules at a first location that is not present in the determined maternal or paternal sequences at the first location; 
 determining, by the computer system, a first fractional concentration of the first sequence in the biological sample at the first location using the sequence reads corresponding to the first location; 
 determining, by the computer system, a second fractional concentration of a second sequence in the biological sample at a second location using the sequence reads corresponding to the second location, the second sequence being inherited by the fetus from the father at the second location, wherein the second sequence is present in the paternal genome at the second location and not present in the maternal genome at the second location; 
 classifying, by the computer system, the first sequence as a de novo mutation at the first location in a fetal genome of the fetus if the first and second fractional concentrations are about the same; and 
 when the first sequence is classified as the de novo mutation:
 outputting, by the computer system, classification information identifying the first sequence as being the de novo mutation at the first location as a result of receiving the sequence reads from the sequencing of the plurality of cell-free nucleic acid molecules from the biological sample obtained from the pregnant female. 
 
 
     
     
       2. The method of  claim 1 , wherein the sequencing of a cell-free nucleic acid molecule is paired-end sequencing that provides a pair of sequences corresponding to both ends of the cell-free nucleic acid molecule, and wherein identifying the location of the cell-free nucleic acid molecule in the human genome includes mapping the pair of sequences to the human genome. 
     
     
       3. The method of  claim 1 , wherein the mapping does not require an exact match of the at least one sequence. 
     
     
       4. The method of  claim 1 , wherein for at least one of the locations, the paternal sequence is determined from a sample of the father. 
     
     
       5. The method of  claim 4 , further comprising:
 performing a random sequencing of a set of cell-free nucleic acid molecules from the sample of the father to obtain sequence reads of the set of cell-free nucleic acid molecules; 
 receiving, at the computer system, the sequence reads of the set of cell-free nucleic acid molecules from the sample of the father; and 
 identifying, by the computer system, a location of each of the set of cell-free nucleic acid molecules in the human genome using the sequence reads of the set of cell-free nucleic acid molecules, thereby determining the paternal sequence for the least one of the locations. 
 
     
     
       6. The method of  claim 1 , wherein for at least one of the locations, the maternal sequence is determined from the biological sample. 
     
     
       7. The method of  claim 1 , wherein identifying the first sequence at the first location includes:
 comparing sequences of cell-free nucleic acid molecules identified to be at the first location to the one or more maternal sequences and the one or more paternal sequences at the first location; and 
 identifying the first sequence of the sequences as not matching any of the one or more maternal sequences and the one or more paternal sequences at the first location. 
 
     
     
       8. The method of  claim 7 , further comprising:
 counting a number of cell-free nucleic acid molecules exhibiting the first sequence; and 
 requiring the number to be greater than a specified number for the first sequence to be considered as a de novo mutation. 
 
     
     
       9. The method of  claim 1 , wherein determining the first fractional concentration includes:
 determining a proportion of the cell-free nucleic acid molecules at the first location that exhibit the first sequence. 
 
     
     
       10. The method of  claim 1 , wherein the second sequence is present on the Y chromosome, or is a genetic polymorphism, or is a single nucleotide polymorphism or is an insertion-deletion polymorphism. 
     
     
       11. The method of  claim 1 , wherein the mother is homozygous for a first allele at the second location and the father is homozygous for a second allele at the second location, the second allele corresponding to the second sequence. 
     
     
       12. The method of  claim 1 , further comprising:
 determining that the first and second fractional concentrations are about the same by:
 computing a difference between the first and second fractional concentrations; and 
 comparing the difference to a cutoff value. 
 
 
     
     
       13. The method of  claim 1 , wherein sequence reads of the plurality of cell-free nucleic acid molecules include at least 187 million sequence reads. 
     
     
       14. The method of  claim 1 , wherein the biological sample includes plasma, serum, urine, saliva, or uterine lavage fluid. 
     
     
       15. The method of  claim 1 , further comprising determining a risk of the fetus suffering from a genetic disease or predisposition to the genetic disease. 
     
     
       16. The method of  claim 15 , wherein the genetic disease is hemophilia A or achondroplasia. 
     
     
       17. A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to identify a de novo mutation in a genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, the father having a paternal genome and the mother having a maternal genome, the instructions comprising:
 receiving sequencing results of a random sequencing of a plurality of cell-free nucleic acid molecules from a biological sample obtained from the pregnant female; 
 identifying a location of each of the plurality of cell-free nucleic acid molecules in a human genome; 
 for each of at least a portion of the locations, determining one or more maternal sequences in the maternal genome and one or more paternal sequences in the paternal genome at the location; 
 identifying a first sequence in the plurality of cell-free nucleic acid molecules at a first location that is not present in the determined maternal or paternal sequences at the first location; 
 determining a first fractional concentration of the first sequence in the biological sample at the first location; 
 determining a second fractional concentration of a second sequence in the biological sample at a second location, the second sequence being inherited by the fetus from the father at the second location, wherein the second sequence is present in the paternal genome at the second location and not present in the maternal genome at the second location; and 
 classifying the first sequence as a de novo mutation at the first location in a fetal genome of the fetus if the first and second fractional concentrations are about the same. 
 
     
     
       18. A method of identifying a de novo mutation in a genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, the father having a paternal genome and the mother having a maternal genome, the method comprising:
 performing a random sequencing of a plurality of cell-free nucleic acid molecules from a biological sample obtained from the pregnant female to obtain sequence reads of the plurality of cell-free nucleic acid molecules; 
 receiving the sequence reads at a computer system; 
 identifying, by the computer system, a location of each of the plurality of cell-free nucleic acid molecules in a human genome using the sequence reads of the plurality of cell-free nucleic acid molecules, wherein identifying the location of a cell-free nucleic acid molecule in the human genome includes mapping at least one sequence of the cell-free nucleic acid molecule to the human genome; 
 for each of at least a portion of the locations, determining, by the computer system, one or more maternal sequences in the maternal genome and one or more paternal sequences in the paternal genome at the location; 
 identifying, by the computer system, a first sequence in the sequence reads of the plurality of cell-free nucleic acid molecules at a first location that is not present in the determined maternal or paternal sequences at the first location; 
 determining, by the computer system, a first fractional concentration of the first sequence in the biological sample at the first location using the sequence reads corresponding to the first location; 
 determining, by the computer system, a second fractional concentration of fetal cell-free nucleic acid molecules in the biological sample at a second location that exhibit a fetal-specific epigenetic status, wherein an epigenetic status of fetal-derived and maternal-derived cell-free nucleic acid molecules in the biological sample are different at the second location; 
 classifying the first sequence as a de novo mutation at the first location in a fetal genome of the fetus if the first and second fractional concentrations are about the same; and 
 when the first sequence is classified as the de novo mutation:
 outputting, by the computer system, classification information identifying the first sequence as being the de novo mutation at the first location as a result of receiving the sequence reads from the sequencing of the plurality of cell-free nucleic acid molecules from the biological sample obtained from the pregnant female. 
 
 
     
     
       19. The method of  claim 18 , wherein for at least one of the locations, the paternal sequence is determined from a sample of the father. 
     
     
       20. The method of  claim 18 , wherein for at least one of the locations, the maternal sequence is determined from the biological sample. 
     
     
       21. The method of  claim 18 , wherein identifying the first sequence at the first location includes:
 comparing sequences of cell-free nucleic acid molecules identified to be at the first location to the one or more maternal sequences and the one or more paternal sequences at the first location; and 
 identifying the first sequence of the sequences as not matching any of the one or more maternal sequences and the one or more paternal sequences at the first location. 
 
     
     
       22. The method of  claim 21 , further comprising:
 counting a number of cell-free nucleic acid molecules exhibiting the first sequence; and 
 requiring the number to be greater than a specified number for the first sequence to be considered as a de novo mutation. 
 
     
     
       23. The method of  claim 18 , wherein determining the second fractional concentration includes:
 determining a proportion of the cell-free nucleic acid molecules at the second location that exhibit the fetal-specific epigenetic status. 
 
     
     
       24. The method of  claim 18 , further comprising:
 determining that the first and second fractional concentrations are about the same by:
 computing a difference between the first and second fractional concentrations; and 
 comparing the difference to a cutoff value. 
 
 
     
     
       25. The method of  claim 18 , where the different epigenetic status is reflected by different DNA methylation patterns. 
     
     
       26. The method of  claim 25 , where the different DNA methylation patterns involve the RAS association domain family 1A (RASSF1A) or the holocarboxylase synthetase (biotin-(proprionyl-Coenzyme A-carboxylase (ATP-hydrolysing)) ligase) (HLCS) gene. 
     
     
       27. The method of  claim 18 , further comprising:
 using an assay to detect nucleic molecules having a fetal-specific epigenetic status at the second location in the human genome. 
 
     
     
       28. A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to method of identifying a de novo mutation in a genome of an unborn fetus of a pregnant female, the fetus having a father and a mother being the pregnant female, the father having a paternal genome and the mother having a maternal genome, the instructions comprising:
 receiving sequencing results of a sequencing of a plurality of nucleic acid molecules from a biological sample obtained from the pregnant female, where the biological sample contains a mixture of cell-free maternal and fetal nucleic acids; 
 identifying a location of each of the plurality of nucleic acid molecules in a human genome; 
 for each of at least a portion of the locations, determining one or more maternal sequences in the maternal genome and one or more paternal sequences in the paternal genome at the location; 
 identifying a first sequence in the plurality of nucleic acid molecules at a first location that is not present in the determined maternal or paternal sequence at the first location; 
 determining a first fractional concentration of the first sequence in the biological sample at the first location; 
 determining a second fractional concentration of fetal nucleic acid molecules in the biological sample at a second location that exhibit a fetal-specific epigenetic status, wherein an epigenetic status of fetal-derived and maternal-derived nucleic acid molecules in the biological sample are different at the second location; and 
 classifying the first sequence as a de novo mutation at the first location in a fetal genome of the fetus if the first and second fractional concentrations are about the same. 
 
     
     
       29. A system comprising:
 the computer product of  claim 17 ; and 
 one or more processors for executing instructions stored on the computer readable medium. 
 
     
     
       30. A system comprising:
 the computer product of  claim 28 ; and 
 one or more processors for executing instructions stored on the computer readable medium.